NF-κB and Angiogenesis Inhibitors from the Aerial ... - ACS Publications

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

NF-κB and Angiogenesis Inhibitors from the Aerial Parts of Chresta martii Marcos Marçal Ferreira Queiroz,† Aymeric Monteillier,†,# Sarah Berndt,†,# Laurence Marcourt,† Eryvelton de Souza Franco,‡ Gilles Carpentier,⊥ Samad Nejad Ebrahimi,∥ Muriel Cuendet,† Vanderlan da Silva Bolzani,§ Maria Bernadete Souza Maia,‡ Emerson Ferreira Queiroz,*,† and Jean-Luc Wolfender† Downloaded via UNIV OF SUSSEX on August 1, 2018 at 15:34:28 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland ‡ Pharmacology of Bioactive Products, Federal University of Pernambuco, UFPE, Postal code 50670-901, Recife, Pernambuco, Brazil § Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais, NuBBE, Instituto de Química, UNESP, 14800-900 Araraquara, São Paulo, Brazil ⊥ Laboratoire CRRET, Faculté des Sciences et Technologie, Université Paris Est Créteil, 94010 Créteil Cedex, France ∥ Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, 1983963113 Tehran, Iran S Supporting Information *

ABSTRACT: The ethyl acetate extract of the aerial parts of Chresta martii showed significant in vitro NF-κB inhibition. Bioactivity-guided isolation was undertaken using HPLC microfractionation to localize the active compounds. Different zones of the HPLC chromatogram were linked to NF-κB inhibition. In parallel to this HPLC-based activity profiling, HPLC-PDA-ESI-MS and UHPLC-TOF-HRMS were used for the early identification of some of the compounds present in the extract and to get a complete phytochemical overview. The isolation of the compounds was performed by high-speed counter-current chromatography and further semipreparative HPLC. Using this approach, 14 compounds were isolated, two of them being new sesquiterpene lactones. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, ECD, NMR, and HRMS. All isolated compounds were evaluated for their inhibitory activity of NF-κB and angiogenesis, and compound 2 showed promising NF-κB inhibition activity with an IC50 of 0.7 μM. The isolated compounds 1, 2, 5, 7, and 8 caused a significant reduction in angiogenesis when evaluated by an original 3D in vitro angiogenesis assay.

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he genus Chresta Vell. ex DC. is distributed in the central highlands of Brazil, but is mainly concentrated in the states of Bahia, Goiás, and Minas Gerais.1 Chresta martii (DC.) H. Rob. (Asteraceae) is a plant found in the Xingó region (caatinga ecosystem) in the northeast region of Brazil and is recognized by the local population as a traditional herb used to treat gastric diseases such as gastric ulcers.2,3 Previous studies have demonstrated significant in vivo protective effects of a C. martii ethyl acetate extract against indomethacin-induced gastric lesions in mice.4 This activity was found to be mediated by alpha-2 adrenoceptor activation, but not by nitric oxide release, opioid receptor activation, or prostaglandin synthesis.5 Recently, the flavonoids chrysoeriol and 3′,4′-dimethoxyluteolin were reported from this plant.6 In the present study, bioactivity-guided fractionation of the ethyl acetate extract of C. martii aerial parts was performed to follow the NF-κB inhibitory activity in vitro. NF-κB is a transcription factor that regulates the inducible expression of a wide range of pro-inflammatory mediators.7 Several articles reported © XXXX American Chemical Society and American Society of Pharmacognosy

the gastroprotective effect of NF-κB inhibitors despite the known gastrotoxicity of some anti-inflammatory drugs.8−10 Therefore, this study was performed to identify tentatively compounds that could explain the in vivo protective effects of C. martii ethyl acetate extract against indomethacin-induced gastric lesions in mice.4 Activity-guided isolation was undertaken using a preliminary coarse HPLC-UV fractionation to localize the active compounds in the crude C. martii extract followed by isolation of the active compounds by high-speed counter-current chromatography (HSCCC) and further semipreparative HPLC-UV. Fourteen compounds were isolated using this approach; two of them are new sesquiterpene lactones (3 and 6). Some compounds exhibited potent NF-κB inhibition properties and could provide a rationale for the traditional use of this medicinal plant to treat gastric diseases such as ulcers.2,3 Received: February 21, 2018

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DOI: 10.1021/acs.jnatprod.8b00161 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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ether (14).31 To the best of our knowledge, with the exception of compounds 9, 12, and 14, the other substances are described herein for the first time in the genus Chresta.32−34 Besides the known compounds isolated, the separation afforded two new sesquiterpene lactones (3 and 6), as described below. The HRESIMS spectrum of compound 3 showed a protonated molecular ion at m/z 397.1502 [M + H]+ (calcd for C19H25O9, 397.1499, Δppm = 0.8). The NMR spectroscopic data of 3 showed a high similarity to those of glaucolide B (8).25 The only difference between these compounds was found to be the presence of two acetyl groups in 3 instead of three in 8. The 1H NMR chemical shift of H-8 (4.02 in 3 and 4.78 in 8) suggested that 3 is deacetylated at C-8. The HMBC correlations from the first acetate methyl group (δH 2.08) to the C-13 methylene (δC 56.2) and its carbonyl at δC 172.0 and between the second acetate (δH 2.05) and only its carbonyl (δC 171.6) confirmed that 3 is acetylated at both C-13 and C-10. The NOESY correlations from CH3-15 to H-6 and H-8 and from CH3-14 to H-8 indicated that the relative configuration should be 4R,5S,6S,8S,10R or 4S,5R,6R,8R,10S. The experimental electronic circular dichroism (ECD) spectrum of 3 showed two negative Cotton effects (CEs) around 320 and 223 nm and a positive CE at 275 nm. A comparison of the time-dependent density functional theory (TDDFT)-calculated spectrum and the experimental data showed good agreement with 4S,5R,6R,8R,10S, while the alternative form revealed a curve with the opposite Cotton effect (Figure 2a). On the basis of these results, 3 was assigned as the new germacranolide-type sesquiterpene 8-deacetylglaucolide B. Compound 6 was isolated as an amorphous solid. The HRESIMS showed a molecular ion at m/z 355.1403 [M + H]+ (calcd for C17H23O8, 355.1393, Δppm = 2.8). The NMR spectroscopic data of 6 were consistent with the presence of a glaucolide-type compound except that a hemiacetal (δC 112.9) was observed at C-1 instead of a ketone. This was supported by the HMBC correlation from the C-14 methyl group (δH 1.56) to this hemiacetal, to the oxygenated quaternary carbon in C-10 (δC 85.9), and to the methylene in C-9 (δC 39.6). A first ring system in the form of an epoxide was clearly evidenced by the HMBC correlation from H-8 (δH 5.43) to C-1. A hydroxy group was observed at δH 5.42 (1H, d, J = 5.4 Hz) that was positioned in C-5 from its scalar coupling with H-5 (δH 3.97), and a second hydroxy group (δH 5.09) that correlated in the COSY spectrum with the methylene H-13 (δH 4.11) was positioned at C-13. A weak 4JCH HMBC correlation between the only acetyl group at δH 1.98 and C-10 supported the location of this acetyl at C-10. Thus, a second epoxide was proposed between C-4 and C-1 to fit with the molecular weight of 6. The NOESY correlations observed (in CDCl3, see Experimental Section) between H-6 and both H-9″ and CH3-15, between CH3-14 and H-9″, between H-8 and H-9′, and between H-5 and CH3-15 allowed the structure to be confirmed and a determination of the relative configuration of 6 (Figure 3). The experimental ECD data showed one positive CE at 240 nm along with two sequential negative CEs at 230 and 212 nm. The TDDFT-calculated ECD spectrum for the 1S,4S,5S,6S,8S,10R stereoisomer showed a good fit with the experimental data with one positive and two negative CEs around 240, 230, and 210 nm, respectively (Figure 2b). Based on these results, 6 was identified as a new germacranolide-type sesquiterpene with the structure shown and has been named chrestin B. In an attempt to identify the compounds that were involved in the NF-κB inhibitory activity of C. martii ethyl acetate extract,

Beside their gastroprotective activity, NF-κB inhibitors are also actively investigated for their cancer chemopreventive and therapeutic potential.11 Overactivation of NF-κB is observed in many cancers and leads to increased cell proliferation, resistance to chemotherapy, and angiogenesis12,13 Because angiogenesis is involved in tumor growth, through increased nutrient supply, and metastasis, its inhibition is particularly interesting in cancer chemoprevention and therapy. Therefore, as a part of our research program aiming at finding bioactive compounds for cancer chemoprevention, the isolated molecules were also tested for their antiangiogen properties using a three-dimensional in vitro assay.14 Consistent with previous reports on sesquiterpene lactones with NF-κB inhibitory and antiangiogen properties,15−18 some of the compounds isolated caused a significant reduction in angiogenesis.

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RESULTS AND DISCUSSION The ethyl acetate extract of the aerial parts of C. martii was submitted to a screening procedure for NF-κB inhibition activity.7 The extract exhibited strong NF-κB inhibition activity with 96% inhibition at 6.7 μg/mL. In the same assay, the reference compound, parthenolide, had an IC50 of 0.7 ± 0.1 μM, suggesting a promising activity of the ethyl acetate extract. To localize rapidly the compounds responsible for this activity, the extract was fractionated by coarse reversed-phase semipreparative HPLC into 12 fractions (Figure 1). The HPLC fractions were submitted to the NF-κB inhibition activity assay at the concentration of 10 μg/mL, and inhibitory activity was correlated to fractions F1, F2, F4, F5, F6, F8, and F12 (Figure 1D). In order to identify compounds present in the active fractions in a preliminary manner, the crude extract was analyzed by HPLC-PDA-ELSD and high-resolution UHPLC-TOF-HRMS for dereplication purposes, and the data were searched against compounds reported in the Asteraceae family and the Chresta genus (Figure 1). The UV-PDA spectra of 1−8 and 11 suggested the presence of sesquiterpene lactones,5 and those of 9, 10, and 12−14 were characteristic for flavonoids (Figure 1).19 Two flavonoids recently isolated from this plant were dereplicated as chrysoeriol (12, tR 38.5 min, m/z 299.0562 [M − H]−), a known NF-κB inhibitor previously isolated from Brucea javanica,20 and luteolin 3′,4′-dimethyl ether (14, tR 42.5 min, m/z 315.0878 [M + H]+).6 The identity of 14 was confirmed by comparison of the retention time with a standard. UV and HRMS data of the remaining compounds were not sufficient for any additional early structure assignments. The ethyl acetate extract (3 g) was fractionated by high-speed counter-current chromatography coupled to ultraviolet detection (HSCCC-UV) with a mixture of n-heptane−EtOAc− MeOH−water (2:3:2:3 v/v) into 68 fractions. This yielded six pure compounds (2, 4, 5, 7, 8) (Figure S1, Supporting Information). Other fractions were further purified by semiprepreparative HPLC. This procedure resulted in the isolation of 14 compounds (1−14). The structure elucidation of the isolated compounds were performed based on NMR spectroscopic and HRMS data. The previously known isolated secondary metabolites were identified as piptocarphol (1),21 piptocarphin D (2),22 1,4-dihydroxy5,8,10,13-tetraacetoxycadin-7 (11)-en-6,12-elide (4), 21 13-deacylglaucolide B (5),23 hirsutolide (7),24 glaucolide B (8),25 luteolin (9),26 quercetin 3-methyl ether (10),27 8-Odeacetyl-8-O-propanoyl glaucolide B (11),28 chrysoeriol (12),29 quercetin 3,3′-dimethyl ether (13),30 and luteolin 3′,4′-dimethyl B



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Figure 1. (A) UV and HRMS data of compounds 1−14. (B and C) HPLC-PDA-ELSD analysis of the ethyl acetate extract of the aerial parts of C. martii. (D) Chromatographic zone exhibiting NF-κB inhibition.

IC50 values in the low micromolar range (ranging from 0.7 μM for 2 to 13.6 μM for 4) (Table 1). Sesquiterpene lactones are well-known for their NF-κB inhibition properties, which have been linked to their ability to alkylate the Cys-38 residue of the p65 subunit, inhibiting its

and therefore partly explain its in vivo gastroprotective effects, the isolated compounds were tested for NF-κB inhibition. Since the isolated flavonoids are already known NF-κB inhibitors, they were not evaluated in the biological assays.20,35−37 Of the sesquiterpene lactones isolated, the activity was evidenced, with C



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Chart 1

Figure 3. NOESY correlations observed for compound 6.

Table 1. NF-κB Inhibition of Selected Active Isolated Compounds

interaction with DNA and thus the transcription induction of the targeted genes.38 Among the sesquiterpene lactones isolated, 2 exhibited the highest potency with the same IC50 value as parthenolide, a natural sesquiterpene lactone isolated from Tanacetum parthenium and used as a positive control. Parthenolide has generated substantial interest in cancer chemoprevention and therapy since its discovery in the 1970s, and a more water-soluble derivative, dimethylaminoparthenolide, has been under phase 1 clinical trial for the treatment of leukemia.39 A wide interest in the sesquiterpene lactone family has led to numerous studies elucidating their mode of action and structure-activity relationships. Of note, a QSAR study conducted on 103 sesquiterpene lactones highlighted the correlation between number of alkylating centers such as α,β-unsaturated carbonyl groups, which would react as Michael-type acceptor units, and the NF-κB inhibitory activity.40 Consistent with those results, three of the most potent sesquiterpene lactones isolated in this work (1, 2, and 7) have an α,β,γ,δ-unsaturated lactone unit, affording two potential Michaeltype acceptor sites that could explain their high potency. This feature is probably not the only relevant structural factor, and the

compound

IC50 (μM)a

1 2 3 4 5 7 8 11 parthenolideb

2.2 ± 0.1 0.7 ± 0.1 7.8 ± 0.5 13.6 ± 2.8 2.4 ± 0.4 1.8 ± 0.2 6.5 ± 0.6 5.1 ± 1.0 0.7 ± 0.1

a Mean ± standard deviations obtained from three independent experiments. bPositive control.

remainder of the functionality present in each case may be of significance. In the process of carcinogenesis, NF-κB activation is closely linked to angiogenesis through the induction of various angiogenic factors such as VEGF.13 For example, parthenolide has been found to inhibit VEGF production, leading to antiangiogenic activity.15 In this context, the NF-κB inhibitors isolated in this work were also tested for their antiangiogenic properties using an optimized 3D in vitro angiogenesis model that mimics in vivo angiogenesis in multiple aspects.14 In this model, human

Figure 2. Comparison of experimental and calculated ECD spectra of compounds 3 (a) and 6 (b) and the two possible stereoisomers for each one. The calculation was achieved with TDDFT at the CAM-B3LYP/6-31** level in MeOH. D



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Figure 4. Effect of C. martii ethyl acetate extract (EACM) and isolated compounds on angiogenesis. (A) Representative pictures of 3D angiogenesis cultures treated with vehicle control (DMSO) and compound 2 at 1 μg/mL for 3 days to follow capillary sprout evolution. (B) Quantification of the angiogenic effect. Results represent the average ± SEM of at least 18 spheres. Each antiangiogenic effect significantly different from vehicle control values was determined by one-way ANOVA, followed by the Dunett multiple comparison test. *p < 0.05; **p < 0.001; ***p < 0.001.

profile has not been thoroughly characterized yet, and further studies to address this issue are needed.42

umbilical vein endothelial cells (HUVECs) were used to sprout from the surface of beads embedded in fibrin gels. For this, HUVECs undergo a complex process that requires several morphological changes to establish tube formation. This process involves degradation of the basement membrane by secreted proteases, sprouting, alignment, proliferation, lumen formation, branching, and anastomosis.14 The activity of the isolated compounds was evaluated by measuring the decrease of total capillary length, anchorage junctions, segments, and extremities around the surface of beads embedded in fibrin gels, which were quantified by using an adapted version of the Angiogenesis Analyzer plug-in (Figure 4). The results obtained indicated that the ethyl acetate extract of C. martii (20 μg/mL), as well as compounds 1, 2, 5, 7, and 8 (1 μg/mL), which were the main constituents of this extract, all caused a significant reduction in angiogenesis, as measured by various parameters including the numbers of total capillary length, anchorage junctions, segments, and extremities (Figure 4). The results suggested that the antiangiogenic activities may occur at various steps of the angiogenic process and are not due to a decrease in metabolic activity as measured by the MTT assay (Figure S2, Supporting Information). Among the sesquiterpene lactones evaluated, compounds 1, 2, and 7 showed the most potent antiangiogenic effects. These were also the most active compounds in inhibiting NF-κB, which is consistent with the well-described implication of NF-κB in angiogenesis.13,15,17,18,41 This work provides a determination of the main constituents of the leaves of C. marti. Since the gastroprotective effect of the ethyl acetate extract of this plant has been linked to its antiinflammatory properties as reflected by a decrease in neutrophil migration,4 the isolation of flavonoids and sesquiterpene lactones possessing strong NF-κB inhibitory activity could provide some support for the use of this plant as an antiulcerogenic remedy.4 In addition, the strong NF-κB inhibition combined with the antiangiogenic activity of some of the sesquiterpene lactones was observed. Even if sesquiterpene lactones are the active constituents of numerous therapeutically used plants, their toxicological

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were measured on a JASCO V-650 spectrophotometer. ECD spectra were acquired with a JASCO J-815 polarimeter. NMR spectroscopic data were recorded on a 500 MHz Varian (Palo Alto, CA, USA) INOVA NMR spectrometer and on a Bruker Avance III 500 MHz NMR spectrometer (Rheinstetten, Germany) equipped with a 5 mm cryogenic DCH (1H/13C) probe. Chemical shifts are reported in parts per million (δ) using the residual CD3OD signal (δH 3.31; δC 49.0) or the DMSO-d6 signal (δH 2.50; δC 39.5) as internal standards for 1H and 13C NMR, respectively, and coupling constants (J) are reported in Hz. Complete assignments were obtained based on 2D NMR experiments (COSY, NOESY, HSQC, and HMBC). HRESIMS data were obtained on a Waters Micromass LCT Premier time-of-flight mass spectrometer with an electrospray ionization (ESI) interface (Waters, Milford, MA, USA). HPLC-PDA-ELSD data were obtained with an Agilent HP 1100 series system consisting of an autosampler, high-pressure mixing pumps, and DAD detector (Agilent Technologies, Santa Clara, CA, USA) connected to an ELSD detector Sedex 85 (Sedere, Oliver, France). HPLC fractionation was performed with an Armen modular spot prep II (Saint-Avé, France) and an X-Bridge RP C18 column (5 μm, 250 × 10 mm, i.d.; Waters). HSCCC was performed using a Talto TBE-300B system (Shanghai, People’s Republic of China) equipped with two LC-10AD HPLC pumps (Shimadzu, Kyoto, Japan), a K-200 UV−vis detector (Knauer, Berlin, Germany), and a Büchi C-660 fraction collector (Büchi, Flawil, Switzerland). Semipreparative HPLC was performed using an Armen modular spot prep II (Saint-Avé, France) and X-Bridge RP C18 columns (5 μm, 100 × 19 mm, i.d.; and 5 μm, 250 × 10 mm, i.d.) (Waters). Plant Material. Aerial parts (leaves and flowers) of C. martii were collected in July 2013 in the Xingó region, Sergipe state, Brazil (longitude −37.940 and latitude ranging from −9.5563 to −9.5548, altitude 130 m). After its authentication by Dr. Nádia Roque (Botany Department of the Biology Institute, Federal University of Bahia, Brazil), a voucher specimen (no. 14602) was deposited in Vale do Acaraú State University herbarium (Sobral, Ceará, Brazil). HPLC-PDA-ESIMS Analysis. HPLC-PDA-ELSD-ESIMS data were obtained with an Agilent HP 1100 series system consisting of an E



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semipreparative HPLC on an X-Bridge RP C18 column (5 μm, 250 × 10 mm, i.d.) with MeOH−H2O (18:82), at a flow rate of 5 mL/min, to yield 1 (11.0 mg). Fraction M (50.6 mg) was purified by semipreparative HPLC on an X-Bridge RP C18 column (5 μm, 250 × 10 mm, i.d.) with MeOH−H2O (33:67), at a flow rate of 5 mL/min, to yield 3 (2.1 mg; tR 22 min). Fraction O (112.2 mg) was purified by semipreparative HPLC on an X-Bridge RP C18 column (5 μm, 100 × 19 mm, i.d.) with MeOH−H2O (39:61), at a flow rate of 10 mL/min, to yield 6 (18.7 mg). Finally fraction O (315 mg) was purified by semipreparative HPLC on an X-Bridge RP C18 column (5 μm, 100 × 19 mm, i.d.) with MeOH−H2O (44:56) as solvent, at a flow rate of 10 mL/min, to yield 9 (3.6 mg), 10 (2.9 mg), 11 (1.4 mg), 12 (8.7 mg), 13 (2.0 mg), and 14 (2.6 mg). 8-Deacetylglaucolide B (3): amorphous, white powder; [α]20D −32.6 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 282 (3.57) nm; ECD (MeOH, c 0.9 mM, 0.1 cm path length) [θ]202 +10851, [θ]224 −21759, [θ]278 +7303, [θ]319 −1944; 1H NMR (CD3OD, 500 MHz) δ 1.55 (3H, s, CH3-15), 1.56 (1H, m, H-3″), 1.60 (3H, s, CH3-14), 2.05 (3H, s, CH310b), 2.08 (3H, s, CH3-13b), 2.15 (1H, m, H-2″), 2.33 (1H, d, J = 15.6 Hz, H-9″), 2.41 (1H, dd, J = 15.6, 7.3 Hz, H-9′), 2.48 (1H, m, H-3′), 3.07 (1H, brd, J = 9.5 Hz, H-5), 3.14 (1H, m, H-2′), 4.02 (1H, d, J = 7.3 Hz, H-8), 4.78 (1H, d, J = 9.5 Hz, H-6), 4.95 (1H, d, J = 12.3 Hz, H-13″), 5.08 (1H, d, J = 12.3 Hz, H-13′); 13C NMR (CD3OD, 126 MHz) δ 18.9 (CH3-14), 20.5 (CH3-13b), 20.6 (CH3-10b), 22.4 (CH3-15), 32.6 (CH2-3), 33.3 (CH2-2), 44.1 (CH2-9), 56.2 (CH2-13), 59.5 (CH-5), 62.4 (C-4), 62.9 (CH-8), 82.2 (CH-6), 86.5 (C-10), 125.3 (C-11), 169.5 (C-7), 171.6 (C-10a), 172.0 (C-13a), 173.8 (C-12), 209.0 (C-1); HRESIMS m/z 397.1502 [M + H]+ (calcd for C19H25O9, 397.1499, Δppm = 0.8). Chrestin B (6): amorphous, white powder; [α]20D −11.3 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 279 (3.20) nm; ECD (MeOH, c 0.9 mM, 0.1 cm path length) [θ]201 +2092, [θ]214 −34079, [θ]232 −10359, [θ]243 +8409; 1H NMR (DMSO-d6, 500 MHz) δ 1.26 (3H, s, CH3-15), 1.56 (3H, s, CH3-14), 1.94 (3H, m, H-2, H-3″), 1.98 (3H, s, CH3-10b), 2.06 (1H, m, H-3′), 2.11 (1H, dd, J = 14.0, 6.5 Hz, H-9″), 2.99 (1H, dd, J = 14.0, 8.8 Hz, 9′), 3.97 (1H, dd, J = 9.2, 5.4 Hz, H-5), 4.11 (2H, m, H-13), 5.03 (1H, dq, J = 9.2, 1.3 Hz, H-6), 5.09 (1H, dd, J = 5.9, 5.0 Hz, OH-13), 5.42 (1H, d, J = 5.4 Hz, OH-5), 5.43 (1H, ddd, J = 8.8, 6.5, 1.3 Hz, H-8); 13C NMR (DMSO-d6, 126 MHz) δ 16.6 (CH3-14), 21.5 (CH3-15, 10b), 27.6 (CH2-2), 37.6 (CH2-3), 39.6 (CH2-9), 52.2 (CH2-13), 68.0 (CH-8), 78.9 (CH-5), 80.6 (CH-6), 82.4 (C-4), 85.9 (C-10), 112.9 (C-1), 127.7 (C-11), 165.1 (C-7), 171.8 (C-12), 169.3 (C-10a); 1H NMR (CDCl3, 500 MHz) δ 1.38 (3H, s, CH3-15), 1.65 (3H, s, CH3-14), 1.98 (1H, m, H-2″), 2.04 (3H, s, CH3-10b), 2.06 (1H, m, H-2′), 2.09 (1H, m, H-3″), 2.13 (1H, dd, J = 14.0, 6.8 Hz, H-9″), 2.29 (1H, dt, J = 12.3, 9.0 Hz, H-3′), 3.14 (1H, dd, J = 14.0, 8.5 Hz, H-9′), 4.21 (1H, d, J = 9.2 Hz, H-5), 4.35 (1H, dd, J = 13.5, 1.7 Hz, H-13″), 4.43 (1H, d, J = 13.5 Hz, H-13′), 5.10 (1H, d, J = 9.2 Hz, H-6), 5.30 (1H, t, J = 8.5, 6.8 Hz, H-8); 13 C NMR (CDCl3, 126 MHz) δ 17.2 (CH3-14), 22.0 (CH3-15), 22.3 (CH3-10b), 28.7 (C-2), 38.7 (C-3), 40.7 (C-9), 55.0 (C-13), 69.0 (C-8), 80.7 (C-5), 81.7 (C-6), 83.1 (C-4), 87.0 (C-10), 114.2 (C-1), 128.1 (C-11), 164.8 (C-7), 170.3 (C-10a), 172.2 (C-12); HRESIMS m/z 355.1403 [M + H]+ (calcd for C17H22O8, 354.1315, Δppm = 2.8). Computational Details. Conformational analyses for compounds 3 and 6 was carried out using MacroModel 9.1 software (Schrödinger, LLC, New York, USA) by applying the OPLS-2005 force field in H2O. The selected conformers were subjected to geometrical optimization using the density function theory (DFT) with the CAM-B3LYP functional and the 6-31G** basis-set as implemented with the Gaussian 09 program package.45 Vibrational analysis was performed at the same level to confirm the stability of the minima. Time-dependent density function theory calculation at the TDDFT/CAM-B3LYP/6-31G** level in MeOH using the “self-consistent reaction field” method with the conductor-like polarizable calculation model was employed to calculate excitation energy (denoted by wavelength in nm) and rotatory strength in dipole velocity (Rvel) and dipole length (Rlen) forms. ECD curves were calculated based on rotatory strengths using a halfbandwidth of 0.3 eV with SpecDis version 1.61.46 Biological Assays. Inhibition of NF-κB Activity. NF-κB inhibitory activity and cell viability were assessed using the HEK293/NF-κB-luc

autosampler, high-pressure mixing pumps, and DAD detector (Agilent Technologies) connected to an ELSD detector Sedex 85 (Sedere, Oliver, France) and to a Finnigan MAT LCQ ion-trap mass spectrometer (Finnigan, San Jose, CA, USA) equipped with a Finnigan electrospray interface (ESI). The HPLC separations were performed on a X-Bridge C18 column (5 μm, 250 × 4.6 mm i.d.). The solvent system used was a mixture of MeOH (A) and H2O (B) in gradient mode: 20% to 70% A in 50 min followed by 70% to 100% A for 10 min; flow rate: 1 mL/min; injection volume: 10 μL (concentration 10 mg/mL in MeOH). The column was used at room temperature (23 °C). The UV absorbance was measured at 210 and 254 nm, and UV spectra (PDA) were recorded between 190 and 600 nm (in increments of 2 nm). ELSD conditions: temperature of 50 °C, gain of 7, and N2 as nebulization gas. ESIMS conditions: capillary voltage 30 V; capillary temperature 200 °C; source voltage 4.5 kV; source current 80 μA; nitrogen as the sheath gas; positive- and negative-ion mode. Spectra (180−1200 amu) were recorded every 0.20 s. UHPLC-TOF-HRMS Analysis. High-resolution mass spectrometry (HRMS) metabolite profiling of the extracts was performed on a Micromass-LCT Premier time of flight (TOF) mass spectrometer (Waters) equipped with an electrospray interface and coupled to an Acquity UPLC system (Waters) using a generic method previously described.43 HPLC-UV Microfractionation of the Crude Ethyl Acetate Extract. The ethyl acetate extract was fractionated using semipreparative HPLC-UV equipment and was performed with an X-Bridge RP C18 column (5 μm, 250 × 10 mm, i.d.; Waters). The flow rate was set at 4 mL/min, and the injection volume was 200 μL (40 mg of the crude extract). The solvent system used was (A) MeOH and (B) H2O; gradient: 20% to 70% A in 50 min followed by 70% to 100% A in 10 min. In order to obtain a coarse fractionation and identify the zone(s) of the chromatogram containing bioactive constituents, the fractions were collected every 10 min. After collection, each fraction was evaporated to dryness using a SpeedVac (HT-4X Genevac, Stone Ridge, NY, USA). The separation yielded 12 fractions: each fraction was analyzed by HPLC-PDA-ELSD with the same conditions used for the analysis of the crude plant extract in order to check the fraction profiles. The fractions were evaluated for their NF-κB inhibition activity. Extraction and Isolation. The air-dried plant material (500 g) was pulverized with a Wiley mill and extracted at room temperature successively with cyclohexane, ethyl acetate, and methanol to give 75, 42, and 48.5 g of residue, respectively. The extracts were concentrated under pressure and later lyophilized. The ethyl acetate extract (2 g) was purified by HSCCC-UV. The biphasic solvent system was selected using the AriZona methodology, which uses a quaternary solvent system composed of n-heptane−EtOAc (upper phase) and MeOH− water (lower phase) with 23 different proportions (fractions A to Z).44 For this purpose, 1 mg of the EtOAc extract of C. martii was used for each system. After thoroughly equilibrating the solvent mixtures in a separatory funnel at room temperature, two phases were collected, separated, dried, and analyzed by HPLC-PDA. The partition coefficient (Kp) was calculated based on the area of each of the peaks detected by UV spectroscopy. As a result, the solvent system selected was n-heptane− ethyl acetate−methanol−water (8.3:41.7:8.3:41.7; v/v/v/v). The coil was first filled with the two phases (upper and lower, 1:1), and rotation was set to 1000 rpm. The lower phase was then pumped into the column at a flow rate of 3 mL/min using the head-to-tail mode (mobile phase = lower phase; stationary phase = upper phase), and the rotation was set at 8000 rpm. After equilibrium between the two phases had been attained, the sample, 2.0 g in 20 mL of a solution with upper and lower phases (1:1), was injected. After the collection of 44 fractions, the rotation was inverted to the tail-to-head mode (mobile phase = upper phase; stationary phase = lower phase). The HSCCC separation yielded 68 fractions in total (F1−F68), which were analyzed by HPLCPDA-ELSD. Six compounds were isolated in one step with the following yields: fraction 28 yielded 2 (9.7 mg), fraction 37 yielded 4 (10.5 mg), fraction 49 yielded 8 (27.7 mg), fraction 55 yielded 7 (11.8 mg), and fraction 63 yielded 5 (7.2 mg). Fractions having similar profiles in the HPLC-PDA-ELSD analysis were combined, resulting in 16 fractions (A−Q) in total. Fractions A, M, N, and O were selected for further purification. Fraction A (83.0 mg) was purified by F



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cell line (RC0014, Panomics, Fremont, CA, USA), as previously described.47 Parthenolide was used as the positive control. MTT Assay. Endothelial cells (1.0 × 104 HUVECS/well) in EBM2 (PromoCell) supplemented with 0.5% fetal bovine serum were added to 96-well plates and then incubated under a fully humidified atmosphere of 95% air and 5% CO2 at 37 °C overnight. The next day, compounds (1 μg/mL) were added to the cells. After 24 h of incubation, MTT (10 μL, 5 mg/mL) was added to the cells for 4 h at 37 °C. Insoluble formazan crystals were dissolved by the addition of 100 μL of DMSO per well. Absorbance was read at 595 nm on a microplate reader (Biotek). Inhibition of Angiogenesis. MTT Assay. Angiogenesis was evaluated by using a modified fibrin gel bead assay as described by Nakatsu et al.14 In this model, the 3D sprouting of HUVECs from the surface of dextran-coated beads embedded in fibrin gels (GE Healthcare) was analyzed. In brief, HUVECs were mixed with Cytodex 3 microcarrier beads at a cell density of 400 HUVECs per bead in a solution of 1000 beads/mL EGM-2 medium. Beads and HUVECs were co-incubated at 37 °C and 5% CO2 and gently shaken every 20 min for 4 h to allow HUVECs to adhere to the bead surface. HUVEC-coated beads were then transferred to a 75 cm2 tissue culture flask and incubated for 24 h. After incubation, HUVEC-coated beads were collected and resuspended at a density of 500 cell-coated beads/mL in a solution of fibrinogen type I (2.5 mg/mL) containing aprotinin (0.15 U/mL). VEGF (10 ng/mL) was added to the bead suspension. Thrombin (0.04 U) was added to each well. A 80 mL amount of fibrinogen type I-aprotinincoated beads solution was gently mixed with the thrombin and allowed to clot for 2 min at room temperature and then at 37 °C and 5% CO2 for 30 min to promote gel formation. A 120 μL amount of ECGM-2 medium containing VEGF (5 ng/mL) was added to each well and equilibrated with the bead-containing gels for 30 min at 37 °C and 5% CO2. Next, 3700 NHDF were added to the top of the gel and allowed to adhere. After 1 h, ECGM-2 was replaced by fresh medium with or without test compounds. Culture medium was replaced after 24 h and then every 48 h. Sprouting appeared between 2 and 3 days of incubation. Cultures were imaged at day 4. In order to quantify the sprouting microvessel network, samples were automatically scanned with a highthroughput imager (IXM, Molecular Device), at 4× magnification at four sites of each well in order to cover the entire surface of the well. Morphometric parameters of the vessel network were measured using a plug-in for the ImageJ software derived from the Angiogenesis Analyzer.48 In brief, the inner areas of spheres were detected by a threshold using the so-called IsoData method after appropriate image pretreatments to correct lightning defaults and optimize the sphere edge visualization. Light field correction and noise removal were performed by using a synthetic flat field and fast Fourier transform band-pass filtering to identify pseudovascular trees. A gradient filter was then applied before segmentation with the “percentile” threshold method. Anchorage junctions between pseudocapillaries and sphere edges were detected together with junctions, extremities, segments, and branches. Segments were defined as lines ended by two junctions or by one junction and one anchorage, and branches as lines linked to one junction and one extremity or one anchorage and an extremity. This quantification was made on one representative experiment out of four.

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Vanderlan da Silva Bolzani: 0000-0001-7019-5825 Emerson Ferreira Queiroz: 0000-0001-9567-1664 Jean-Luc Wolfender: 0000-0002-0125-952X Author Contributions #

A. Monteillier and S. Berndt contributed equally to the work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the São Paulo State Research Foundation (FAPESP), Coordenaçaõ de Aperfeiçoamento de Pessoal de ́ Nivel Superior (CAPES), and National Research Council (CNPq) for funding and fellowships (2010/09780-0 to M.M.F.Q.). We are very thankful to M. Pupier (Chemistry Department, University of Geneva) for the recording of some NMR data of compound 6 in DMSO-d6.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00161.

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REFERENCES

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Figures S1 to S17 showing the HSCCC-UV chromatogram, MTT biological assay data, and NMR spectra of compounds 3 and 6 (PDF)

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